A traditional dark-field illumination system is used to observe an un-stained semi-transparent material (such as cells, micro-algae or other biological samples) in a transparent medium. The main technology of the dark-field illumination system is based on the principle that scattered and reflective light will be generated when a light passes through a sample substance, and thus the system can only receive the scattered light and a portion of the reflective light by changing the relative angle and position of an observer and a light detection device. Thus, the scattered light and reflective light provide information-rich signals including the profile, the structural variation and the refraction variation.
A dark-field condenser is a common accessory used in a microscope system, wherein the dark-field condenser is co-axially disposed on a light path to generate a hollow-cone illumination for observing micro-particles, micro-pore defect structures or other to-be-observed micro-structures in a transparent medium. A traditional dark-field condenser comprises a positive lens and a circular optical stop. Further, the dark-field condenser can further comprise a reflective mirror or a curved mirror, in order to increase the numerical aperture (N.A.). However, although the foregoing co-axial dark-field focusing method can change the light path by various manners, most of the light path is an annular light field.
For example, U.S. Pat. No. 6,259,557 disclosed a traditional device and method for dark field illumination, as shown in FIG. 1, wherein the device comprises: a light source 11 to provide light, two light collection lenses 12a, 12b to collect the light, a pin-hole stop 13 having a pin-hole 131 to form a light source spot, a field lens 14 through which the light source spot passes to form parallel light, a ring diaphragm 15 having a ring slit 151 through which the parallel light passes, a ring-shape reflection mirror 16 to reflect the parallel light, a fly-eye optical device 17 through which the reflected parallel light passes to form even annular light, and a ring-shape condenser lens 18 through which the annular light passes to form a spot which focuses on a focal plane 19 of an objective lens(not-shown). The purpose of the dark field illumination is to evenly emit light onto the surface of the sample, and to provide higher brightness and contrast image for satisfying needs of observing reflective samples. The focused spot still forms a relatively large excitation region of a diameter about 4-8 mm on the focal plane 19 of the objective lens.
However, because the traditional device of dark field illumination is generally used to satisfy needs of large excitation region and high numerical aperture for image observation, the excitation spot focused by the traditional device is greater than the size scale of 100 micrometer (μm). Thus, when the focal plane 19 is located within a microfluidic channel of a microfluidic chip and the width of the microfluidic channel and samples therein are smaller than 100 μm, the excitation spot focused by the traditional device will be greater than the width of the microfluidic channel and samples therein. As a result, the excitation spot may illuminate inner walls of the microfluidic channel to cause a large number of background noise due to the scattering of the channel wall, so that the traditional device cannot provide a suitable dark-field illumination for clearly observing the samples in the microfluidic channel.
Therefore, it is necessary to provide an objective-type dark-field illumination device for a microfluidic channel to solve the foregoing problems, as described above.